JP4304318B2 - Gene enrichment method - Google Patents

Abstract

The present invention provides a method where it is possible to concentrate the gene which is expressed only in small amount from a mixture of the gene being expressed in large amount and the gene being expressed only in small amount even when the gene being expressed in large amount is unknown.

Description

〔試験例１ 各核酸画分中の遺伝子の検索〕
沈殿をTEに溶解し、その溶液の一部を加えて25μl PCR反応液（2.5μl 10 x PCR Buffer、2μl dNTP、0.25μl rTaq、1μlプライマーセット、テンプレートDNA、H₂Oを加えて25μlとする）を調製し、ミネラルオイルを一滴加えて密閉し、94℃１分間、60℃１分間、72℃２分間のＰＣＲを３５サイクル行い、72℃で8分間静置し、23℃まで降温した。プライマーセットはそれぞれ0.5μlの、EfとEr（大腸菌遺伝子用）、BfとBr（枯草菌遺伝子用）、HfとHr（ヒト遺伝子用）を混ぜたものを用いた。
以下のそれぞれのオリゴヌクレオチドをTEに溶解して100 μM溶液をつくり、上記プライマーとして用いた。
（ａ）大腸菌：ompA ecompa.gb_bal, 1−2271 CDS 172−669の以下の部分をプライマーとした。
Ef 1102-1119: 5' TCCGAAAGATAACACCTG 3'（配列番号１）
Er 1892-1908: 5' GGGATACCTTTGGAGAT 3'（配列番号２）
ＰＣＲによる増幅産物は807塩基対となる。
（ｂ）枯草菌：xynA bpxyna.gb_bal, 1−1070 CDS 61−747の以下の部分をプライマーとする。
Bf 243-260: 5' ATTTAGTGCAGGCTGGAA 3'（配列番号３）
Br 650-672: 5' CGTTTCATACATTTTCCCCATTG 3'（配列番号４）
ＰＣＲによる増幅産物は430塩基対となる。
（ｃ）ヒト：IL5h j03478.gb_pr2, 1−3230の以下の部分をプライマーとした。
Hf 1600-1629: 5' ACTTTTTGAAAATTTTATCTTAATATGTGG 3'（配列番号５）
Hr 1981-2007: 5' TGGCCGTCAATGTATTTCTTTATTAAG 3'（配列番号６）
ＰＣＲによる増幅産物は408塩基対となる。
その他溶液としては、以下のものを用いた。
（ｉ）10 x PCR Buffer: 100mM Tris-HCl, pH8.3 +500mM KCl + 15mM MgCl₂
（ii）dNTP mix: dATP + dGTP + dCTP + dTTP 各2.5mM
（iii）Taq: 5 units/ μl (溶媒：20mM Tris-HCl + 100mM KCl + 0.1mM EDTA + 1mM DTT + 0.5% Tween 20 + 0.5% Nonidet P-40 + 50% グリセロール（Glycerol））
（iv）DNAテンプレート
〔結果１〕
図４Aに見るようにE/B DNA混合液の大腸菌遺伝子と枯草菌遺伝子のPCR産物のバンドはそれぞれのDNAの量をよく反映していた。これをハイブリダイゼーションに1回（1⁰）かけると、大腸菌遺伝子と枯草菌遺伝子がほぼ同量になった。さらに2回（2⁰）、3回（3⁰）かけると、大腸菌遺伝子<<枯草菌遺伝子となった（図５A)。即ち、大部分を占めていた大腸菌DNAが優先的に除去された。これは相対的濃縮度は10,000倍を遥かに越えるものである事を示していた。
図４Bに見るようにH/E/B DNA混合液のヒト遺伝子、大腸菌遺伝子、枯草菌遺伝子のPCR産物のバンドはそれぞれのDNAの量をよく反映していた。枯草菌の遺伝子はDNA混合物の中で非常に少量しか存在しないので、枯草菌DNAのみが極微量存在する場合と比較して、非特異的なバンドが多く、特異的な430塩基対のバンドは僅かに観察された。これをハイブリダイゼーションに3回（3⁰）かけると、ヒト遺伝子と比較して、大腸菌遺伝子が顕著に濃縮されることがわかった（図５B）。コントロールPCR（図４B）との比較により、ヒト遺伝子は数１０分の１、大腸菌遺伝子は5〜10倍であるから、おおよそ数100倍濃縮されたと算定された。枯草菌遺伝子も非特異的なバンドが消え、特異的なバンドがはっきりと同定出来るようになり、莢雑DNAに比して大幅な濃縮が達成されたことが分かる。この時、大腸菌遺伝子と比較しては、特に濃縮が顕著でないことは、最初過剰に存在していたヒトDNAが特異的に除去された事を支持するものと解釈できる。
実施例２
稀少微生物として、好熱好酸性細菌であるSulfolobus shibataeを選び、S. shibataeのＤＮＡと大腸菌のＤＮＡを混合して、実施例１と同様の実験を行い、大腸菌ＤＮＡが選択的に除去されることにより、大腸菌DNAに対して千分の１しか存在しないS. shibataeのDNAが相対的に濃縮されることを確認した。S. shibataeのＤＮＡは菌体より、Ｇｅｎとるくん（商品名 宝酒造株式会社製）を用いて調製した。
なお、実施例２においては実施例１と同様の制限酵素、緩衝液、試薬などを用いたが、実施例１に記載していないものとして以下のようなものを用いた。
(a) 10 x Mirus Labeling Buffer A (Mirus社) : 200mM MOPS, pH7.5
(b) グリコーゲン : 20mg/ml
(c) MAGNOTEX−SA（宝酒造株式会社） :ハイブリダイズしたＤＮＡはMAGNOTEX−SAキットを用い、本キットのマニュアルに記載の方法で除去した。 すなわち、MAGNOTEX−SA 50μl（1mg）を1．5mlチューブに入れ、チューブを磁気スタンドで1分間静置後、上清を除去した。ビオチン標識DNA溶液と等量の本キットに添付の2 x Binding Bufferを混合し、チューブに加えて混和後、室温で10分間静置した。チューブを磁気スタンドに１分間静置後、上清を回収した。さらに1 x Binding Buffer 200μlをMAGNOTEX−SAに添加し洗浄し、洗浄をもう1回繰り返した。混和後、チューブを磁気スタンドに１分間静置後、上清を回収した。
〔操作１ DNA混合液の作製〕
80μgの大腸菌DNAと80ngのS.shibatae DNAを440μｌのTEに溶解して、A液とした。
〔操作２ ドライバーDNAの調製（DNAの断片化とビオチン化）〕
75μgの大腸菌DNAと75ｎｇのS.shibatae DNAを含むＡ液に、120μｌの10 x B、 600単位のMsp Iと水を加えて1.2mlにして37℃で2時間保温してDNAを切断し、次いで60℃、15分間の処理でMspIを失活させた。反応液を3等分し、DNA断片をエタノール沈殿させ、遠心分離機にかけ回収した（以下、これをE/SミックスＤＮＡ断片と呼ぶ）。
各試験管中の沈殿（それぞれ１/３等量のE/SミックスＤＮＡ断片を含む）を、200μｌの水+25μｌの10 x Mirus Labeling BufferAに溶かし、25μｌのMirus Label IT試薬を加えて37℃、2時間反応させ、ＤＮＡをビオチンで標識した。25μｌの5M NaCl、550μｌのエタノールを加え−20℃で保存した。
〔操作３ ターゲットＤＮＡの調製（ＤＮＡの部分分解）〕
2.5μgのS.shibatae DNAを含むA液に、10μlの10 x M、10μlの0.1% BSA、20.4単位のSse 8387Iと水を加え100μlとして、37℃で2時間保温してＤＮＡを切断し、次いで60℃、15分間の処理でSse 8387Iを失活させた。10μgのNaOAc、220μｌのエタノール、1μｌのグリコーゲンを加えて−20℃で保存した（以下、これをE/SターゲットDNA断片と呼ぶ）。
〔操作４ ハイブリダイゼーション〕
E/SターゲットＤＮＡのエタノール沈殿を含む試験管を15000ｒｐｍ x 20分間の遠心にかけ、上清を捨てた。同じ試験管にE/SドライバーＤＮＡのエタノール沈殿の懸濁液400μｌを加え、15000ｒｐｍ x 20分間の遠心にかけた。上清を捨て、さらに425μｌのE/SドライバーＤＮＡのエタノール沈殿の懸濁液を加えて遠心し、上清を捨てた。最後の沈殿を室温で9.28μｌの水＋0.96μｌの3Ｎ NaOHに溶かし、氷上で急冷し、0.96μｌの3N HCl＋50mM Tris-HCl, pH 7.2を加え、次いで8μｌの20 x SSC、20.8μｌのホルムアミドを加えて良く混合し、これを0⁰とした。１滴のミネラルオイルを滴下し、蓋で密閉して、37℃で保温してハイブリダイゼーション反応を進行させた。24時間後、ハイブリダイゼーション反応液の10μｌを取り出し、1mgのMAGNOTEX−SＡによりビオチン標識ＤＮＡの除去を行った。回収した上清に20μｌのNaOAc、500μｌの−20℃のエタノール、1μｌのグリコーゲンを加え、15000ｒｐｍ x 15分間の遠心で沈殿を得て、これを1⁰とした。
また、残りのハイブリダイゼーション反応液の10μｌ（2⁰）、20μｌ（3⁰）それぞれについてもMAGNOTEX−SＡによりビオチン標識ＤＮＡの除去を行った。回収した上清に20μｌのNaOAc、500μｌの−20℃のエタノール、1μｌのグリコーゲンを加え、15000ｒｐｍ x 15分間の遠心で沈殿を得た（2⁰沈殿：A、3⁰沈殿：B）。一方、1/3当量（2⁰）および1本分（3⁰）のドライバーＤＮＡを同条件にて遠心し沈殿を分離し、80 μｌの水 +10 μｌの 3N NaOHに溶解した後、その全量でA、Bそれぞれを溶解した。15μｌの3N HCl+50mM Tris-HCl pH 7.2を加えてから300μｌの−20℃のエタノールを加え混合して、−80℃の冷蔵庫に30分間以上静置した。遠心によって得た沈殿に4.64 μｌの水+0.48μｌ 3N NaOHを加え室温で十分に溶かし、氷上で急冷してから順次0.48μｌの3N HCl+50mM Tris-HCl pH 7.2、4μｌの20 x SSC、10.4μｌのホルムアミドを加え、1滴のミネラルオイルを重層して、37℃に保温した。24時間後、Aの試験管からハイブリダイゼーション反応液の10μｌを取り出し、先と同様の手順によりターゲットDNAの回収を行い、2⁰とした。
Bについても同様の手順でビオチン標識DNAの除去、上清の回収し、15000rpm x 15分間の遠心で沈殿を得た（3⁰沈殿：C）。1/3当量のドライバーDNAを遠心し沈殿を分離し、50 μｌの水 +10 μｌ 3N NaOHに溶解した後、その全量でCを溶解した。10μｌの3N HCl+50mM Tris pH 7.2を加えてから300μｌの−20℃のエタノールを加え混合して、−80℃の冷蔵庫に50分間以上静置した。遠心によって得た沈殿に4.64 μｌの水 +0.48 μｌ 3N NaOHを加え室温で十分に溶かし、氷上で急冷してから順次0.48μｌの3N HCl+50mM Tris-HCl pH 7.2、4μｌの20 x SSC、10.4μｌのホルムアミドを加え、1滴のミネラルオイルを重層して、37℃に保温した。24時間後、Cの試験管からハイブリダイゼーション反応液10μｌを取り出し、同様の手順によりターゲットDNAの回収を行い、3⁰とした。最後に、すべての回収されたＤＮＡの沈殿を50μｌの1 x TEに溶解した。
〔試験例１ ＤＮＡ画分中の遺伝子の検索〕
25μｌの PCR反応液（2.5μｌ 10 x PCR Buffer，2μｌ dNTP，0.25μｌ rTaq，1μｌプライマーセット，3μｌテンプレートDNA，H₂Oを加えて25μｌとする）を調製し、Thermal cycler（宝酒造株式会社）により、94℃1分間、60℃1分間、72℃2分間のPCRを35サイクル行い、72℃で8分間静置し、4℃まで降温した。プライマーセットはそれぞれ0.5μｌの、EfとEr（E.coli遺伝子用）、SfとSr（S.shibatae遺伝子用）を用いた。
以下のそれぞれのオリゴヌクレオチドをTEに溶解して100μM溶液をつくり、上記プライマーとして用いた。
（a）大腸菌 ：ompA 遺伝子（ecompa.gb_bal）の以下の部分をプライマーとした。
Ef2 5’-TCCGAAAGATAACACCTG-3’（配列番号７）
Er2 5’-GGGATACCTTTGGAGAT-3’（配列番号８）
PCRによる増幅産物は８０７塩基対となる。
（b）S.shibatae ：エステラーゼ遺伝子（EstI）の以下の部分をプライマーとした。
Sf 5’-ATGCCCCTAGATCCTCGAATC-3’（配列番号９）
Sr 5’-TCAACTTTTATCATAAAATGTACG-3’（配列番号１０）
PCRによる増幅産物は９１８塩基対となる。
その他溶液としては、以下のものを用いた。
（i）10 x PCR Buffer： 100mM Tris-HCl，pH8.3 + 500mM KCl + 15mM MgCl₂
（ii）dNTPmix： dATP + dGTP + dCTP + dTTP 各2.5ｍM
（iii）Taq： 5 units/μｌ（溶媒：20mM Tris-HCl + 100mM KCl + 0.1mM EDTA + 1mM DTT + 0.5%Tween20 + 0.5%Nonidet P-40 + 50%グリセロール）
（iv）DNAテンプレート
〔試験例２ リアルタイムPCR法による定量〕
DNA混合液中の各遺伝子の存在比を解析するため，リアルタイムPCR法により，サイクル毎の増幅の様子を解析した。上記のＰＣＲ法では各遺伝子の存在比を解析することは不可能であるため、LightCyclerクイックシステム330（ロッシュ・ダイアグノスティックス社製）を用いて大腸菌遺伝子およびS.shibatae遺伝子の存在比を解析した。
ＰＣＲ反応液組成は、5μｌ テンプレートDNA、2μｌ LightCycler−DNAマスターSYBR GreenI、1μｌ プライマーセット、1.6μｌ 25mM MgCl₂（終濃度3mM）で、水を加えて20μｌとした。
以下のそれぞれのオリゴヌクレオチドをTEに溶解して10μM溶液をつくり、上記プライマーとして用いた。
（a）大腸菌 ：ompA 遺伝子（ecompa.gb_bal）の以下の部分をプライマーとした。
Ef2 5’-TCCGAAAGATAACACCTG-3’（配列番号７）
Er2 5’-GGGATACCTTTGGAGAT-3’（配列番号８）
PCRによる増幅産物は８０７塩基対となる。
（b）S.shibatae ：エステラーゼ遺伝子（EstI）の以下の部分をプライマーとした。
Sf 5’-ATGCCCCTAGATCCTCGAATC-3’（配列番号９）
Sr 5’-TCAACTTTTATCATAAAATGTACG-3’（配列番号１０）
PCRによる増幅産物は９１８塩基対となる。
〔結果２〕
図７に示すように、大腸菌遺伝子のバンドは濃縮するに従ってしだいに減少しており、3⁰においては完全に除去されていることが分かる。一方、S.shibatae遺伝子は一連の行程を通じてほぼ同量であることが確認できる（なお、低分子側のバンドはプライマーがダイマーになったものと考えられる）。全体的に非特異的バンドが多いのはターゲットDNAをSau3A1で部分消化したためと考えられる。
E．coli DNAの増幅曲線はターゲットDNAが18.2サイクル、1⁰DNAが20.74サイクル、2⁰DNAが23.31サイクル、3⁰DNAが23.80サイクルにおいて指数関数増殖期に入っており、このことからコピー数はターゲットDNAがもっとも多く、1⁰、2⁰、3⁰の順に減少しており、大部分を占めていたE.coli DNAが優先的に除去されたことが分かる。
一方、S.shibatae DNAの増幅曲線はターゲットDNA、1⁰DNAが19.22サイクル、2⁰DNAが20.58サイクル、3⁰DNAともに20.65サイクルとなっており，1⁰→2⁰において減少がみられるものの、大腸菌の減少率と比較すると、一連の濃縮過程を経た後でも存在比はほとんど変化していないといえる。また、２回のハイブリダイゼーションによって、E.coli DNAとS.shibatae DNAはほぼ同量になり、さらに３回のハイブリダイゼーションによって、E.coli DNA<<S.shibatae DNAとなった。以上の結果から、リアルタイムPCR法を用いてDNA混合液中の各遺伝子の存在比を解析することによって、図７の電気泳動の結果に示されているE.coli DNAが選択的に除去されることが、定量的に確認された。
産業上の利用可能性
本発明に係る微量遺伝子の濃縮方法、微量遺伝子の濃縮用キットまたは微量遺伝子濃縮装置を用いれば、ＤＮＡ試料中の微量遺伝子を多量遺伝子よりも多量となるまで濃縮することができる。しかも、ＤＮＡ試料中の除去すべき多量遺伝子が既知である必要もない。したがって、例えば土壌、湖水または河水などの自然界の標品から抽出したＤＮＡ等を試料として用いることができるという利点がある。
このように微量遺伝子を濃縮することにより、その遺伝子をクローニングをする為にスクリーニングするべき遺伝子ライブラリーの総クローン数を大幅に減少させることができる。その結果、人的労力、費用および時間の大幅な削減を可能とする。したがって、化学工業的に重要な酵素の遺伝子または医薬・農薬のリード化合物、抗生物質の合成遺伝子群などの有用遺伝子を従来の方法よりも容易かつ迅速に取得できるようになる。そのほかにも、本発明に係る微量遺伝子の濃縮方法などを用いて細胞表面抗原の遺伝子を取得して、その遺伝子ないし遺伝子情報を用いて抗体を作製して、その抗原を持つ細胞ないし微生物を同定し、また例えばＦＡＣＳのようなセルソーターを用いて該細胞ないし微生物を単離することもできる。さらに、この単離した細胞ないし微生物の全ゲノムの塩基配列を解析する事により新たに有用な遺伝子を取得できる。また、ヒトを含む動植物に寄生または感染している微生物の遺伝子を本発明に係る方法または装置などで濃縮することにより、寄生・感染微生物の同定を可能とし、さらに、その遺伝子の機能を明らかにして利用することを可能とするなどの多種多様な応用が可能である。
【配列表】

【図面の簡単な説明】
第１図は、ゲノムサイズとＣｏｔ_１／２との関係を示す。より具体的には、種々の二本鎖核酸の再形成を示す。ゲノムサイズは図上部にヌクレオチド対として示した（新生化学実験講座、核酸Ｉ ２００頁）。
第２図は、仔ウシ胸腺ＤＮＡの再形成を示す。△（白抜き三角）は仔ウシ胸腺ＤＮＡの濃度が２μｇ／ｍｌの試料を表し、●は仔ウシ胸腺ＤＮＡの濃度が１０μｇ／ｍｌの試料を表し、○は仔ウシ胸腺ＤＮＡの濃度が６００μｇ／ｍｌの試料を表し、△（塗りつぶし三角）は仔ウシ胸腺ＤＮＡの濃度が８，６００μｇ／ｍｌの試料を表し、＋は放射線標識大腸菌ＤＮＡ４３μｇ／ｍｌを内部標準として８，６００μｇ／ｍｌの仔ウシ胸腺ＤＮＡを加えた試料を表す（新生化学実験講座、核酸Ｉ １９９頁）。
第３図は、実施例の操作の流れを表す。
第４図（Ａ）は、Ｅ／ＢターゲットＤＮＡ Ｓｓｅ８３８７Ｉ処理画分４μｌをＰＣＲによりそれぞれの遺伝子を増幅し、１０μｌを常法により、１００ボルト、４０分間、３％アガロースゲル電気泳動にかけ、エチジュームブロマイドで染色したときの電気泳動ゲルを示す。レーン１：大腸菌遺伝子、レーン２：枯草菌遺伝子、レーン３：酵母遺伝子（参考）、レーン４：サイズマーカーＤＮＡ（下より１００、２００、３００、４００、５００、６００、７００、８００、９００、１，０００、１，２００、１，５００塩基対。以下同じ。）
第４図（Ｂ）は、Ｈ／Ｅ／ＢターゲットＤＮＡ Ｓｓｅ８３８７Ｉ処理画分４μｌをＰＣＲによりそれぞれの遺伝子を増幅し、１０μｌを常法により、１００ボルト、４０分間、３％アガロースゲル電気泳動にかけ、エチジュームブロマイドで染色したときの電気泳動ゲルを示す。レーン１：サイズマーカーＤＮＡ、レーン２：ヒト遺伝子、レーン３：大腸菌遺伝子、レーン４：枯草菌遺伝子
第５図（Ａ）は、アルカリ処理しエタノール沈殿させた核酸を１８μｌ水＋１μｌ ＴＥに溶解、１０ｘＰＣＲ Ｂｕｆｆｅｒ、Ｂｘ１０、ｄＮＴＰ、ｒＴａｑ、プライマーセットを加えて２５μｌの反応液とし、ＰＣＲを行い、反応液の一部を常法により、１００ボルト、４０分間、３％アガロースゲル電気泳動にかけ、エチジュームブロマイドで染色したときの電気泳動ゲルを示す。レーン１，２，４，５，７，８は３μＬ、レーン３，６は９μＬの反応液の一部を用いた。レーン１：１^０大腸菌遺伝子、レーン２：１^０枯草菌遺伝子、レーン３：２^０大腸菌遺伝子、レーン４：２^０大腸菌遺伝子、レーン５：２^０枯草菌遺伝子、レーン６：３^０大腸菌遺伝子、レーン７：３^０大腸菌遺伝子、レーン８：３^０枯草菌遺伝子、レーン９：サイズマーカーＤＮＡ
第５図（Ｂ）は、アルカリ処理しエタノール沈殿させた核酸を１８μｌ水＋１μｌ ＴＥに溶解、１０ｘＰＣＲ Ｂｕｆｆｅｒ、Ｂｘ１０、ｄＮＴＰ、ｒＴａｑ、プライマーセットを加えて２５μｌの反応液とし、ＰＣＲを行い、当量の反応液の一部を常法により、１００ボルト、４０分間、３％アガロースゲル電気泳動にかけ、エチジュームブロマイドで染色したときの電気泳動ゲルを示す。レーン１：サイズマーカーＤＮＡ、レーン２：３^０ヒト遺伝子、レーン３：３^０大腸菌遺伝子、レーン４：３^０枯草菌遺伝子
第６図 本発明に係る微量遺伝子濃縮方法の一態様における操作の流れを示す。なお、点線は、多量遺伝子の除去状態を検出する場合の操作の流れを示す。
第７図は、実施例２の試験例１の結果を示す。ターゲットＤＮＡ（０^０）、１^０、２^０、３^０をＰＣＲによりそれぞれの遺伝子を増幅した。８μｌを、１００Ｖ、３０分間、０．８％アガロース（株式会社ニッポンジーン製）で電気泳動にかけ、エチジウムブロマイド染色した。サイズマーカーにはλ−Ｈｉｎｄ ＩＩＩ ｄｉｇｅｓｔを用いた。レーン１：サイズマーカーＤＮＡ（λ−Ｈｉｎｄ ＩＩＩ）、レーン２：０^０大腸菌遺伝子、レーン３：０^０Ｓ．ｓｈｉｂａｔａｅ遺伝子、レーン４：１^０大腸菌遺伝子、レーン５：１^０Ｓ．ｓｈｉｂａｔａｅ遺伝子、レーン６：２^０大腸菌遺伝子、レーン７：２^０Ｓ．ｓｈｉｂａｔａｅ遺伝子、レーン８：３^０大腸菌遺伝子、レーン９：３^０Ｓ．ｓｈｉｂａｔａｅ遺伝子、レーン１０：大腸菌のテンプレートＤＮＡを含まないＰＣＲ反応液、レーン１１：Ｓ．ｓｈｉｂａｔａｅのテンプレートＤＮＡを含まないＰＣＲ反応液Technical field
The present invention relates to a method for concentrating trace genes derived from rare microorganisms or microorganisms that cannot be cultured in a DNA sample prepared from a mixture of microorganisms, or trace genes that are expressed only in trace amounts in animal and plant cells, and the concentration The present invention relates to a trace gene obtained by the method, a method for analyzing the trace gene, an apparatus for concentrating the trace gene, and a kit for enriching the trace gene.
In this specification, a gene that was initially present in a trace amount in a DNA sample is referred to as a “trace gene”, and a gene that was initially present in a large amount is referred to as a “multiple gene”.
Background art
Although useful substances produced by microorganisms are widely used as industrial enzymes and antibiotics, 95 to 99% of microorganisms existing in nature cannot be cultured, and these microorganisms that cannot be cultured are now It is not used industrially. However, it acquires genes of industrially useful enzymes from microorganisms that cannot be cultured, or acquires biosynthetic genes of useful substances produced by microorganisms such as antibiotics and expresses them in appropriate hosts Then, since useful substances such as new industrial enzymes and antibiotics can be produced in large quantities, their industrial utility is immeasurable.
In order to obtain a useful gene as described above from a microorganism, usually, a microorganism having a useful gene is isolated and cultured, and DNA is extracted from the microorganism to prepare a gene library. In principle, the gene library may be introduced and expressed in an appropriate host, and transformants having activity may be screened and selected by an appropriate method. However, this method requires a great deal of time and effort to obtain useful genes from microorganisms that occupy only a part of the mixed preparation of various microorganisms or microorganisms that cannot be isolated and cultured. Cost. In particular, when the proportion of a microorganism having a useful gene is very small, and the microorganism is a microorganism that cannot be cultured, it is practically impossible to obtain the useful gene from the microorganism. Become. If it is possible to relatively concentrate genes derived from microorganisms that are present in a small amount in microorganism preparations and microorganisms that cannot be cultured in preparations, that is, trace genes or few genes can be relatively concentrated, subsequent screening of useful genes can be performed. Efficient and saves labor, time and money.
On the other hand, the cells that make up the human body are divided into more than 200 types, each cell has a common genome with about 100,000 genes, and expresses tens of thousands of genes depending on the cell type. It is believed that Examining the expression of such genes is becoming more and more important not only for obtaining knowledge about the function of individual genes but also for elucidating life phenomena. Moreover, as a result of detailed analyzes on a small number of relatively limited genes so far, it has been found that many genes work cooperatively in many life phenomena.
Gene expression is roughly divided into three classes based on the expression level. About 10 per cell^3-4Abundant class of copy grade, about 10²Copy intermediate class, and about 10¹The Rare class has only a copy level. On the other hand, regarding the types of expressed genes, there are tens of thousands of types per cell in mammals, and most genes belong to the Rare class. That is, the expressed gene in the cell has a high expression level (10^3-4(Copy) Abundant class genes are present in very few species and expressed in very small amounts (10¹(Copy) There are numerous types of Rare class genes (for example, Alberts, B., et al. (1989) Molecular biology of the cell, 2nd edition. Garland Publishing Inc.). Under such circumstances, the need for a technique for analyzing a large number of genes, including trace genes, in more detail is expressed, and using these analyzes is also expected to be used for medical purposes such as genetic diagnosis. .
Currently, canonical gene analysis includes canonicalization (Minoru SHKo (1990) Nucleic Acids Res., 18, 5705-5711), differential display (Liang, P., and Pardee, AB (1992) Science 257 , 967-971) or molecular index method (Kikuya Kato (1995) Nucleic Acids Res., 23,3685-3690) etc.) and other methods based on multiplex PCR and methods using DNA chips are known. ing. In the canonicalization method, a small amount of a gene is classified as a large amount of gene by placing a polymer nucleic acid mixture under hybridization conditions and separating the nucleic acid that has become double-stranded and the one that remains in a single strand at an appropriate time. It can be concentrated to the same amount. However, in the canonicalization method, the amount of the trace gene after the treatment cannot be larger than that of the large amount of gene, so the effect of concentration is limited. When analysis using multiplex PCR such as differential display method is performed, competitive PCR reaction occurs, and a strong bias is applied to the abundance of the gene, so the detection sensitivity is lower than normal PCR, and the Rare class gene Is known to be difficult to detect (David J. Bertioli, et al. (1995) Nucleic Acids Res., 23, 4520-4523). As a method for overcoming such problems, Japanese Patent Application Laid-Open No. 2000-37193 discloses a method for removing a large number of known genes present in a nucleic acid sample and concentrating a small amount of genes. Otherwise, this method cannot be used.
On the other hand, as a method for identifying a mutant gene of a certain species, there is a so-called subtraction method and its modified method (Ellen E. Lamar and E. Palmer (1984) Cell, 37, 171-177, Ilse Wieland et al. (1990 Proc. Natl. Acad. Sci. USA, 87, 2720-2724, Anne Kallioniemi et al. (1992) Science 258, 818-821, Nikolai Lisitsyn et al. (1993) Science 259, 946-951). These are genes that are qualitatively or quantitatively different among the same genes (A and B) in two individuals of the same species, or mRNA in the same type of cells in two different states. A method for concentrating and isolating genes (referred to as A and B) having different abundances in cDNA prepared from the method, wherein the gene is identical to B (A) in A (B) by some method The principle is that a specific gene that exists only in A (B) can be obtained by removing B using the gene of B (A). Therefore, the method inevitably requires two types of DNA samples in which most of the contained genes are the same.
However, there are very many types of microorganisms in natural samples (soil, lake water, river water, etc.), and their microbial composition generally differs depending on the sample, and each has its own composition. . Therefore, when enriching trace genes derived from rare microorganisms in it, it is impossible to prepare two types of DNA samples that are the same in most of the included genes, one to be pulled and one to be pulled. The trace gene cannot be concentrated using the subtraction method. In addition, since a small amount of genes in a cDNA sample are concentrated, most of the contained genes are the same, and it is extremely difficult to prepare two types of cDNA samples: a cDNA sample containing the trace gene and a cDNA sample not containing it. Therefore, it is difficult to concentrate the trace gene by the subtraction method.
  Disclosure of the invention
The present invention provides a method for concentrating trace genes derived from rare or non-culturable microorganisms that are present only in a small amount in a specimen, and among a large number of genes expressed in individual organisms, biological tissues or cells. An object of the present invention is to provide a method capable of concentrating a trace gene even if it is unknown about the gene, and an apparatus and kit for enriching the trace gene.
As a result of extensive research, the inventor found that in the Cot analysis, which has been used for genome size analysis and repetitive sequence analysis, the double-stranded remodeling reaction rate is inversely proportional to the genome size and proportional to the concentration of the same sequence. From this, it was conceived that trace genes and trace genes in a DNA sample containing a large amount of genes could be concentrated, and it was confirmed that trace genes in the mixed solution could be concentrated from a mixed solution of E. coli DNA, Bacillus subtilis DNA and calf thymus DNA. Thus, the present invention has been completed.
That is, the present invention
(1) A DNA sample containing a gene that is present in a trace amount and a gene that is present in a large amount is subjected to the following operation to separate a gene that is present in a trace amount from a gene that is present in a large amount. Gene enrichment method,
(A) Divide the DNA sample into 2 minutes. One DNA sample is a driver DNA fraction and the other DNA sample is a target DNA fraction.
(B) The target DNA and the driver DNA are mixed, and the DNA in the mixed solution is made into a single strand. Alternatively, the target DNA and driver DNA are mixed into a single strand.
(C) Hybridization is performed, and the double-stranded DNA formed by the driver DNA and the target DNA is removed from the mixed solution.
(D) The operations of (b) and (c) are performed one or more times using the DNA solution obtained by removing the double-stranded DNA obtained in (c) instead of the target DNA.
(2) A DNA sample containing a gene present in a small amount and a gene present in a large amount is subjected to the following operation to separate a gene present in a small amount from a gene present in a large amount. Gene enrichment method,
(A) Divide the DNA sample into 2 minutes. One DNA sample is a driver DNA fraction and the other DNA sample is a target DNA fraction.
(B) The DNA is cleaved in each of the driver DNA fraction and the target DNA fraction. However, at this time, the molecular weight of the driver DNA is set to be lower than that of the target DNA.
(C) Label the driver DNA. If desired, a linker / adapter is attached to the target DNA.
(D) The target DNA and an excessive amount of labeled driver DNA are mixed, the DNA in the mixed solution is made into a single strand, and then hybridization is performed.
(E) The double-stranded DNA formed by the driver DNA and the target DNA is removed from the mixed solution by labeling the driver DNA.
(F) The operations of (d) and (e) are performed one or more times using the DNA solution obtained by removing the double-stranded DNA obtained in (e) instead of the target DNA.
(3) In the above (1) or (2), the ratio d / t between the mixing amount (d) of the driver DNA and the mixing amount (t) of the target DNA is more than 1 and 1000 or less. A method for concentrating the gene present in a trace amount,
(4) The method for concentrating a gene present in a trace amount according to (2) or (3), wherein the driver DNA is labeled with biotin, digoxin, fluorescein or rhodamine,
(5) The driver DNA has an average strand length of 200 to 300 base pairs, and the target DNA has an average strand length of 1000 base pairs or more, and is present in a trace amount as described in (2) to (4) above Gene enrichment method,
(6) The DNA is cleaved by a 4-base recognition restriction enzyme in the driver DNA fraction and by a 5-8 base recognition restriction enzyme in the target DNA fraction, as described in (2) to (5) above A method for concentrating genes present in trace amounts of
(7) The method for concentrating a gene present in a trace amount as described in (6) above, wherein the DNA is cleaved by Msp I in the driver DNA fraction and Sse 8387I in the target DNA fraction,
(8) The method for concentrating a gene present in a trace amount according to (2) to (5), wherein the DNA is cleaved by ultrasonic waves or mechanical shearing force,
(9) A DNA sample containing a gene that is present in a trace amount and a gene that is present in a large amount is subjected to the following operation to separate a gene that is present in a trace amount from a gene that is present in a large amount. Gene enrichment method,
(A) Divide the DNA sample into 2 minutes. One DNA sample is a driver DNA fraction and the other DNA sample is a target DNA fraction.
(B) The DNA is cleaved in each of the driver DNA fraction and the target DNA fraction. However, at this time, the molecular weight of the driver DNA is set to be lower than that of the target DNA. If desired, a linker adapter is attached to the target DNA.
(C) In each of the driver DNA fraction and the target DNA fraction, the DNA is made into a single strand.
(D) Immobilize single-stranded driver DNA on a carrier.
(E) A carrier on which a single-stranded driver DNA is immobilized is brought into contact with or mixed with a single-stranded target DNA solution to perform hybridization.
(F) By separating the carrier and the solution, the target DNA that forms a double strand with the driver DNA is removed.
(G) The operations of (e) and (f) are performed one or more times using the target DNA solution obtained in (f) instead of the target DNA solution.
(10) A DNA sample is prepared from a DNA sample extracted from a sample in which at least two types of microorganisms, organisms, biological tissues or cells are mixed, or nucleic acids extracted from individual organisms, biological tissues or cells and / or nucleic acids thereof. A method for concentrating a gene present in a trace amount as described in (1) to (9) above,
(11) The abundance ratio of a gene having a large abundance before the treatment of a trace gene having a small abundance before the treatment is increased after the treatment, as described in (1) to (10) above DNA sample obtained by the method.
(12) A step of concentrating a gene present in a trace amount (hereinafter referred to as “trace gene”) by the method described in (1) to (10) above, and a trace gene is obtained from a DNA sample in which the obtained trace gene is concentrated. A method of analyzing a trace gene, characterized by comprising a step of obtaining and analyzing a base sequence of the trace gene;
(13) a gene obtained from a DNA sample in which a small amount of the gene obtained by the method according to (1) to (10) is concentrated,
(14) (a) means for single-strand DNA in a mixed solution of target DNA and labeled driver DNA; (b) means for performing hybridization; and (c) driver DNA by means of labeling of driver DNA. Means for removing the double-stranded DNA formed by the target DNA, and (d) using the DNA solution from which the double-stranded DNA obtained in (c) has been removed, instead of the target DNA, A trace gene concentrator characterized by comprising means for repeating the means of (c),
(15) (a) a means for fixing the single-stranded driver DNA to the carrier; and (b) contacting or mixing the single-stranded driver DNA-immobilized carrier with the single-stranded target DNA solution. And (c) means for removing the target DNA that has formed double strands with the driver DNA by separating the carrier and the solution, and (d) instead of the solution of the target DNA (c And a means for repeating the means of (b) and (c) using the solution of the target DNA obtained in (1),
(16) A means for cleaving DNA, a labeling substance or a carrier, a reagent for labeling DNA or a reagent for immobilizing DNA on a carrier, a hybridization reagent, and a double-stranded DNA of driver DNA and target DNA A kit for concentrating trace genes, characterized by comprising means for removing,
About.
  BEST MODE FOR CARRYING OUT THE INVENTION
The present invention will be described in detail below. First, the Cot analysis that became the theoretical background of the present invention (Koji Sawada et al., New Chemistry Laboratory, Nucleic Acid I, Chapter 10 Nucleic Acid Fractionation by Hybridization, 193-238) Page).
The hybridization reaction of the polymer nucleic acid follows the following secondary reaction formula (1) in which effective collisions between complementary strand molecules are rate-limiting, and the reaction rate is proportional to the square of the concentration [C] of the single-stranded DNA.
d [C] / dt = −k [C]²         (1)
(In the formula, [C] represents the concentration of single-stranded DNA, t represents the reaction time, and k represents the reaction rate constant.)
When a repetitive sequence is included, such as DNA of animal cells, the initial concentration varies depending on the sequence, so that the reaction progress is multiphasic. When one of the complementary strands is in a large excess (20 times or more), it can be handled as a pseudo primary reaction.
The primary structure of nucleic acids can be analyzed based on the kinetic considerations of nucleic acid hybridization reactions in the liquid phase. This is the Cot / Crt analysis. Cot analysis is DNA-DNA hybridization, Crt analysis is DNA-RNA or RNA-RNA hybridization analysis, and the latter is also called Rot analysis. In 1968 Britten and Kohne (R. J. Britten and D. E. Kohne, Science,161, 529) kinetically analyze the double-stranded DNA remodeling reaction of animal cell DNA, (1) analysis of genome size (variety of genomic DNA base sequences), (2) analysis of repeated base sequences First, it was shown that Cot analysis can be used. Cot and Crt analysis is also used as an index for fractionation of nucleic acids.
(1) Cot analysis and genome size
Formula (1) is integrated to obtain the initial concentration [C₀]
[C] / [C₀] = 1 / (1 + k [C₀T) (2)
(Where [C₀] Represents the initial concentration of single-stranded DNA. [C], t and k are the same as defined above. )
[C₀] [C] / [C on the horizontal axis₀] Is plotted on the vertical axis to obtain a Cot curve (FIG. 1).
As the unit of Cot, nucleotide mol · sec / l is used. When the average molecular weight of nucleotide is 314, 1 μg / mL DNA is 3.19 × 10^-6Since it becomes nucleotide mol / l,
[C₀] T = DNA (μg / ml) × 1.15 × 10^-2 × Time (hr) (3)
It becomes. Approximating this,
[C₀] T = DNA (A260) × time (hr) / 2 (4)
(In the formula, A260 represents absorbance at 260 nm.)
When there are no repetitive sequences in the DNA molecule, the Cot curve becomes a first order sigmoid curve (FIG. 1).
From (2)
([C₀]-[C]) / [C] = k [C₀] T (5)
(Where [C₀], [C], t and k are the same as defined above. )
[C₀] T on the horizontal axis, ([C₀]-[C]) / [C] is plotted on the vertical axis to form a straight line. The reciprocal of the gradient is [C₀] T_1/2It is.
[C₀] T_1/2= 1 / k (6)
(Where t_1/2Represents the time when 50% of the single-stranded DNA becomes double-stranded DNA. [C₀] And k are the same as defined above. )
When the DNA concentration is aligned with the nucleotide mol concentration, the double-stranded remodeling reaction rate is inversely proportional to the genome size, and thus [C₀] T_1/2Is proportional to From this, the genome sizes of various organisms can be calculated (FIG. 1).
(2) Repeat (repeat) sequence
When there are repetitive sequences in the DNA molecule, the Cot curve is a synthesis of a primary sigmoid curve of a unique sequence and a primary sigmoid curve of each repetitive sequence. In the case of calf thymus DNA, the reaction is divided into two stages, about 40% [C₀] T_1/2The value is 0.03, 60% is [C₀] T_1/2It is approximated as a composite of primary sigmoid curves with a value of 3000. The latter is a unique sequence, and the former is a highly repetitive sequence with 100,000 copies (FIG. 2). It is possible to separate the unique sequence and the repetitive sequence by stopping the reaction at an appropriate time and separating single-stranded and double-stranded DNA.
Here, for example, DNA extracted from a standard mixture of at least two types of microorganisms such as soil, lake water, river water, organisms, biological tissues, or cells can be considered equivalent to the genome of one higher organism, Defined as metagenome. In the metagenome, a gene derived from a minimal amount of microorganisms or the like can be regarded as a unique sequence, and a gene derived from a microorganism or the like that exists more than that can be regarded as a repeated sequence. The repetitive sequence is classified as a highly repetitive sequence, a moderate repetitive sequence, or a low repetitive sequence according to the number of the repetitive sequences.
In addition, nucleic acids extracted from individual organisms, biological tissues or cells and / or DNA prepared from the nucleic acids have a great diversity in their abundance, as are cDNAs prepared from mRNA, and are classified as unique sequences and repetitive sequences. Is possible.
Based on the above theory of Cot analysis, DNA with a larger copy number is faster to be double-stranded. Therefore, after the DNA sample used in the present invention is made into a single strand under appropriate conditions, the double strand is reformed. Then, if the formed double-stranded DNA is separated and removed by an appropriate method, trace genes are relatively concentrated in the remaining single-stranded DNA sample. However, it should be noted here that enrichment in this case is only relative, and trace genes can never become majority, and at most, at most as much as high-volume genes It is only that amount. As mentioned above, this is the method actually used as a canonical method.
    However, the inventors have conceived that the abundance of a trace gene can be increased after treatment by a method as described below, and completed the present invention.
Specifically, the gene enrichment method according to the present invention comprises concentrating trace genes by separating a trace amount gene from the large amount gene by subjecting a DNA sample containing the trace gene and the large amount gene to the following operation. Features. (A) Divide the DNA sample into 2 minutes. One DNA sample is a driver DNA fraction and the other DNA sample is a target DNA fraction. (B) The target DNA and the driver DNA are mixed, and the DNA in the mixed solution is made into a single strand. Alternatively, the target DNA and driver DNA are mixed into a single strand. (C) Hybridization is performed, and the double-stranded DNA formed by the driver DNA and the target DNA is removed from the mixed solution. (D) The operations of (b) and (c) are performed one or more times using the DNA solution obtained by removing the double-stranded DNA obtained in (c) instead of the target DNA.
The principle will be described below. Here, a gene having a large amount is referred to as gene A, and a gene having a small amount is referred to as gene B. A unique sequence including the partial sequence of gene A and / or its peripheral sequence in the driver DNA is A, and B is a specific sequence including the partial sequence of gene B and / or its peripheral sequence. A and B have the same sequence in the target DNA fragment. [Xⁿ] Represents the concentration of X when n times of hybrid / non-hybrid separation operations were performed. “= ˜” indicates the same amount or the same amount.
In the first hybridization and the hybrid / non-hybrid separation operation, A is largely removed, and its concentration is [target A¹] ([Target A⁰]> [Target A¹]). On the other hand, [Target A⁰+ Driver A⁰] >> [Target B⁰+ Driver B⁰Therefore, if Cot is appropriate, [Target B¹] Has almost no change. At the next hybridization, [Target A¹+ Driver A⁰] = ~ [Driver A⁰] >> [Target B¹+ Driver B⁰Therefore, the reaction is a pseudo-primary reaction that depends almost exclusively on the concentration of the driver DNA. That is, compared with A, hybridization of B is almost negligible, and after the hybrid / non-hybrid separation operation, target A is further removed [target A²] ([Target A¹]> [Target A²]) [Target B²] Has almost no change. By repeating this, [Target Aⁿ] << [Target Bⁿ] = ~ [Target B⁰Thus, only the gene B having B remains in the target DNA.
In other words, the microgene concentration method according to the present invention is characterized in that the driver A newly supplied at each hybridization is always highly concentrated in the hybridization system, and therefore the hybridization rate of A is outstanding. It is fast. In the conventional canonicalization method, if the concentrations of A and B are approximately the same, the probability of hybridization of each becomes equal, and therefore one cannot be preferentially concentrated. However, in the present invention, since the concentration of the driver A is always remarkably high, it is possible to concentrate the gene B to a larger amount than the gene A as follows.
(I) When [Target A] >> [Target B] (for the first operation)
In this case, since there is a large amount of driver A in the hybridization system in which target DNA and driver DNA are mixed, the probability that driver A effectively collides with target A and hybridizes is much higher than in the case of B. By selectively removing the double-stranded DNA formed by the driver DNA and the target DNA, the target A is efficiently removed, while the target B is hardly removed and remains in the system. Therefore, gene A (a large amount gene) and gene B (a trace gene) can be separated and gene B (a trace gene) can be concentrated.
(Ii) [Target A] = ˜ [Target B] (when the operation is repeated)
Concentration was repeated, and [Target Aⁿ] And [Target B]ⁿ] In the new hybridization system in which this solution and the driver DNA are mixed, the concentration of the newly supplied driver A is remarkably high, so that the target A and the driver A are effective. The probability of collision is much higher than the probability that target B and driver B collide effectively. Therefore, the target A hybridized with the driver A is removed, and most of the target B remains in the system. Therefore, the gene B (trace gene) can be concentrated until the amount is higher than that of the gene A (large gene).
    The DNA sample used in the present invention is not particularly limited and may be any sample. For example, a DNA sample extracted from a sample in which at least two or more kinds of microorganisms, organisms, biological tissues or cells are mixed can be mentioned. Therefore, DNA extracted from the sample can be used without separating and culturing the microorganism from a sample having a unique microbial composition obtained in nature such as soil, lake water, river water and the like. The method for concentrating trace genes according to the present invention has an advantage that it can be applied to a sample having such a unique microbial composition.
The DNA sample used in the present invention may be a nucleic acid extracted from an individual organism, a biological tissue or a cell and / or a DNA sample prepared from the nucleic acid. For example, DNA extracted from a mixture of inflamed tissue and normal tissue, a mixture of cancerous cells and normal cells, or a mixture of cells infected with microorganisms, viruses, etc. and non-infected cells And / or cDNA samples prepared from mRNA. Further, it may be a cDNA sample prepared from mRNA in which many types of genes are expressed in cells.
As a DNA extraction method, for example, a method known per se such as an alkaline SDS method, a centrifugal method, or a combination thereof may be used. Commercially available DNA extraction kits can be used as appropriate.
    As one embodiment of the present invention, (1a) a DNA sample is divided into two (one DNA sample is a driver DNA fraction, and the other DNA sample is a target DNA fraction); (1b) target DNA and driver DNA is mixed and the DNA in the mixed solution is made into a single strand, or the target DNA and the driver DNA are made into a single strand and then mixed, and (1c) hybridization is performed, and the driver DNA and the target are mixed. (1d) and (1b) and (1) using the DNA solution from which the double-stranded DNA obtained in (1c) is removed instead of the target DNA. Examples of the method include separating the trace gene from the large amount gene and concentrating the trace gene by performing the operation 1c) one or more times.
In the above operation (1a), the DNA sample may be arbitrarily divided into two. One DNA sample is a driver DNA fraction and the other DNA sample is a target DNA fraction. However, when there is only a small amount of DNA sample, it is preferable to divide into two so that the driver DNA fraction is larger in view of the mixing ratio of the target DNA and driver DNA in the operation (1b). Furthermore, when the total amount of DNA is not sufficient, after amplification by PCR in advance, the target DNA fraction and the driver DNA fraction may be separated and the subsequent operations may be performed. Here, when only the driver DNA fraction is amplified by the PCR method, the difference in the degree of amplification depending on the sequence gives a complicated bias to the sequence to be concentrated later.
In the operation (1b), it is preferable to mix an excessive amount of driver DNA with respect to the target DNA. This is because a large amount of genes can be efficiently removed by adding an excessive amount of driver DNA. More specifically, the ratio d / t between the mixing amount (d) of the driver DNA and the mixing amount (t) of the target DNA exceeds 1 and is about 1000 or less, preferably about 10 to 1000, more preferably About 100-1000 is suitable. In particular, the hybridization time can be shortened when the amount of driver DNA mixed is large. However, if the mixing ratio of driver DNA is increased, a larger amount of DNA sample is required. Therefore, in the present invention, when there are not enough DNA samples, the mixing ratio d / t is more than about 1 and preferably about 10, more preferably about 10.
As a method for making DNA into a single strand, a method known per se may be used. For example, it can be made into a single strand by applying heat at about 94 ° C. for about 1 minute or by alkali treatment. However, the conditions described in the examples are preferred. Further, the target DNA and the driver DNA may be mixed and then single-stranded, or the target DNA and driver DNA may be single-stranded before mixing.
In the operation (1c), the target DNA and the driver DNA are hybridized. The hybridization conditions at this time may follow a method known per se, but the conditions described in the Examples are preferred. Here, the hybridization of the target DNA and the driver DNA includes a state where the driver DNA is hybridized to a part of the target DNA, or a state where a plurality of driver DNAs are hybridized to the target DNA.
Subsequently, the double-stranded DNA in which the target DNA and the driver DNA are hybridized is removed. At the same time, double-stranded DNA in which driver DNAs are hybridized and single-stranded driver DNA may also be removed. As a method for removing the double-stranded DNA hybridized with the target DNA and the driver DNA, a method known per se may be used. For example, a batch method using hydroxyapatite or a column chromatography method, CsCl or the like is used. Examples thereof include a specific gravity centrifugation method.
By the above operation (1c), a large amount of genes are removed and a small amount of genes are concentrated. However, in order to sufficiently remove a large amount of genes, using the DNA solution obtained by removing the double-stranded DNA obtained in step (1d), ie, (1c), instead of the target DNA, (1b) and (1c) The operation is preferably performed, and at least the operation (1d) is more preferably repeated at least once.
    In a preferred embodiment of the present invention, (2a) the DNA sample is divided into two (one DNA sample is the driver DNA fraction and the other DNA sample is the target DNA fraction), and (2b) the molecular weight of the driver DNA is Cleave the DNA in each of the driver DNA fraction and the target DNA fraction so as to have a low molecular weight relative to the target DNA, (2c) label the driver DNA, and optionally attach a linker adapter to the target DNA; (2d) The target DNA and an excessive amount of labeled driver DNA are mixed, the DNA in the mixed solution is made into a single strand, and then hybridization is performed. (2e) The driver DNA is labeled with the driver DNA. Remove double-stranded DNA formed by the target DNA from the mixed solution . At this time, double-stranded DNA formed by driver DNAs and single-stranded driver DNA are also removed. Then, instead of (2f) the target DNA, using the DNA solution from which the double-stranded DNA obtained in (2e) has been removed, the steps (2d) and (2e) are carried out one or more times to obtain a trace gene. There is a method in which a small amount of genes are concentrated by separating from a large amount of genes.
In the operation (2b), as a method for cleaving DNA, a method known per se can be used, and specifically, restriction enzyme treatment, ultrasonic treatment, physical shearing force or the like can be used. When cleaving using a restriction enzyme, it can be cleaved into a DNA fragment of a desired size by using a restriction enzyme with a different number of base pairs to be recognized. For example, when 8-base recognition Not I is used, the expected fragment length is an average of 65,536 base pairs, which is about 60 bacterial genes long. If a shorter one is desired, for example, using Cpo I with 7 base recognition, one base pair is A or T.⁶X2 = 8,192 base pairs is the expected average length, including 7-8 genes.
In general, if the target DNA is enlarged, a plurality of genes can be obtained. Accordingly, the target DNA preferably has an average chain length of about 1000 base pairs or more. Since a gene related to a certain metabolic system often forms a gene cluster composed of a plurality of such genes, the method according to the present invention is useful for obtaining a cluster gene. At this time, the size of the driver DNA may be the same as that of the target DNA, but a smaller one is preferable because efficiency and specificity of hybridization are increased. Specifically, it may be about 200 to 300 base pairs, which is the size (chain length) of DNA normally used for Cot analysis. If the size (strand length) of the DNA is too long, the composition of the sequence on single-stranded DNA, such as that some parts have similar sequences and some parts are unique, makes the above size preferable.
Thus, in order to make the molecular weight of the driver DNA lower than that of the target DNA, the driver DNA fraction is operated with a 4-base recognition restriction enzyme, and the target DNA fraction is operated with a 5-8 base recognition restriction enzyme. It is preferable to cleave the DNA in (3b). It is more preferable to cleave the DNA with Msp I that recognizes 4 bases in the driver DNA fraction and Sse 8387I that recognizes 8 bases in the target DNA fraction.
The driver DNA is preferably labeled in advance before hybridization (operation (2c)). By labeling, there is an advantage that the double-stranded DNA hybridized with the driver DNA and the target DNA can be efficiently removed. For labeling the driver DNA, any known one can be used as long as it can separate labeled DNA and unlabeled DNA. In addition to avidin used in the examples, for example, digoxin, fluorescein, rhodamine and the like can be used.
The double-stranded DNA formed by the driver DNA and the target DNA can be removed from the mixed solution (operation (2e)) according to a method known per se according to the label. For example, when biotin is used as a label, double-stranded DNA in which target DNA and driver DNA are hybridized with dynabeads bound with avidin can be removed. Moreover, a method using an anti-labeled antibody can also be mentioned.
In addition, after the target DNA is fragmented, a linker / adapter may be added (operation (2c)). By doing so, amplification by the PCR method and integration into a cloning vector can be facilitated after the concentration operation.
The other operations are the same as the above operations (1a) to (1d).
    As another preferred embodiment of the present invention, (3a) the DNA sample is divided into two (one DNA sample is the driver DNA fraction and the other DNA sample is the target DNA fraction), and (3b) the driver DNA DNA is cut in each of the driver DNA fraction and the target DNA fraction so that the molecular weight is lower than that of the target DNA, and a linker / adapter is attached to the target DNA as desired, and (3c) the driver DNA fraction and In each of the target DNA fractions, the DNA is single-stranded, (3d) the single-stranded driver DNA is fixed to the carrier, and (3e) the single-stranded driver DNA-immobilized carrier is single-stranded. Contact or mix with the solution of the target DNA thus prepared, perform hybridization, and then (3f) carrier (3e) Using the target DNA solution obtained in (3f) instead of the target DNA solution, (3e) And (3f) are performed one or more times to separate a trace gene from a large amount of gene and concentrate the trace gene.
Instead of labeling the driver DNA, the driver DNA is fixed to the carrier in advance before hybridization in order to efficiently remove the double-stranded DNA hybridized with the driver DNA and the target DNA. Is also preferable (operation (3c)). As a method for fixing the driver DNA to the carrier, a method known per se can be used. For example, a method of adsorbing on a nitrocellulose membrane or nylon membrane, a method of physically adsorbing on a glass surface coated with polylysine or a glass surface treated with silane, and the like can be mentioned. Another example is a method of immobilizing the biotin-labeled driver DNA on a carrier on which streptavidin is immobilized.
By fixing the driver DNA to the carrier in this way, by separating the solution of the carrier and the target DNA, double-stranded DNA formed by the driver DNA and the target DNA from the target DNA solution, that is, a large amount of gene Most can be easily removed (operation (3f)).
Here, in order to remove a large amount of genes with high accuracy, it is also effective to remove while detecting the removal state of the large amount of genes. Therefore, in the present invention, for example, the degree of removal of a large amount of genes is monitored using a removed double-stranded DNA solution or using a recovered single-stranded DNA solution. A method of performing the removing operation (2f) or (3g) may be used. Such a method may be combined with a method known per se, but a specific embodiment is shown in FIG. The process is the same as described above until the hybridized double-stranded DNA is removed. Using the removed double-stranded DNA solution, a large amount of genes therein are detected using a method known per se. As a result, for example, if a large amount of gene is not substantially detected, or if the amount is sufficiently reduced compared to the concentration of the large amount of gene in the double-stranded DNA solution initially removed, Therefore, it is not necessary to repeat the operation (2f) or (3g), and it is possible to move to a trace gene acquisition step. On the other hand, when the opposite result is obtained, the above operation (2f) or (3g) is preferably performed.
    In order to monitor the degree of removal of such abundant genes, the amount of the abundant genes can be estimated using a sequence (common sequence) common to the genomes of biological species present in large quantities as a marker. In order to measure the abundance of the common sequence, a hybridization method using the common sequence as a probe or a PCR method using the common sequence as a primer can be used. As a common sequence, in the case of DNA extracted from a sample in which many kinds of organisms are mixed, a common sequence present in a gene (rDNA) encoding rRNA is desirable. Since the base sequence of rDNA is known for many biological species, it is easy to find the common sequence of rDNA, and the large number of copies of rDNA per genome is suitable as a marker for the common sequence in rRNA. That is why. When the species present in large quantities are limited, for example when limited to prokaryotes, there are more common sequences. Further, when the species is limited, for example when it is limited to actinomycetes, there are many more common sequences. The common sequence need not be limited to rDNA, and may be a common sequence in the genome of a biological species present in a large amount, for example, a common sequence present in a gene encoding a protein that is commonly present in the biological species. . For example, since the cytochrome C gene is present in all living species and the nucleotide sequences are known for many living species, the common sequence present in the cytochrome C gene is suitable as a marker.
When a large amount of genes in a DNA sample are derived from biological solids, biological tissues / cells of the same species, a gene specific to the species can be used as a marker for estimating the amount of the large amount of genes. When the DNA sample is a cDNA derived from a biological solid or biological tissue / cell of the same biological species, a gene with a high expression level can be used as a marker.
The number of repetitions of this operation (2f) or (3g) is not uniquely determined, and in consideration of the Cot value, each recovered DNA fraction obtained by operating once, twice,... N times. It is preferable to use a plurality of recovered DNA fractions for gene acquisition. This is because there is a possibility that useful genes can be obtained from a plurality of DNA fractions having different concentrations of microorganisms / organisms having different numbers in the first sample. As the operation is repeated, the smallest amount of genes can be obtained more easily.
    In a more preferred embodiment of the present invention, (4a) a DNA sample is divided into two (one DNA sample is a driver DNA fraction and the other DNA sample is a target DNA fraction), and (4b) a restriction enzyme is used. The target DNA fraction is cleaved into long DNA (target DNA) having an average strand length of about 1000 base pairs or more, and one is cleaved into short DNA (driver DNA) having an average strand length of about 200 to 300 base pairs. (4c) The driver DNA is chemically biotinylated. (4d) The biotinylated driver DNA (the mixing amount is d) and the target DNA (the mixing amount is t) have a mixing ratio d / t exceeding 1 and not more than 1000, preferably 10 After mixing to a certain degree, the DNA in the mixed solution is made into a single strand, and then hybridized in a liquid phase. (4e) Add avidin dynabeads to the mixed solution and use it to remove double-stranded DNA formed by hybridizing the driver DNA and target DNA. That is, biotin attached to the driver DNA forms a complex with avidin. Therefore, the target DNA hybridized with the driver DNA is captured by the dynabead. Since the dyna beads can be easily removed by centrifugation, the double-stranded DNA formed by hybridizing the driver DNA and the target DNA together with the dyna beads can also be easily removed. At this time, double-stranded DNA formed by driver DNAs and single-stranded driver DNA are also removed. (4f) The method of performing the operation of (4d) and (4e) one or more times using the supernatant obtained in (4e) instead of the target DNA can be mentioned.
    By using the gene enrichment method according to the present invention as described above, a DNA sample characterized in that the abundance ratio of a trace amount gene to a large amount of gene before treatment increases after treatment can be obtained. Trace genes can be obtained from such DNA samples. Upon acquisition, for example, a method known per se such as the method described in Molecular Cloning, 2nd ed., Cold Spring Harbor Laboratory (1989) is used. Good. Commercially available kits can also be used. In this case, if the adapter / linker sequence added is used, it can be easily cloned by a method known per se. The obtained trace gene can be decoded as it is, or can be inserted into an appropriate vector and subcloned, and then the nucleotide sequence can be decoded. Decoding of the base sequence may be performed using a method known per se based on the Maxam-Gibert method or the Sangar method, or a commercially available DNA sequencer may be used.
    The present invention also provides a kit for concentrating trace genes in the DNA sample. Specifically, a means for cleaving DNA, a labeling substance or carrier, a reagent for labeling DNA or a reagent for immobilizing DNA on a carrier, a reagent for hybridization, and driver DNA and target DNA A kit having a means for removing the double-stranded DNA formed by and is preferred. Here, the labeling substance or carrier is used for labeling the driver DNA or immobilizing the driver DNA in order to easily remove the double-stranded DNA formed by hybridizing the driver DNA and the target DNA. The “reagent for labeling DNA” is a reagent used for labeling driver DNA, and the “reagent for immobilizing DNA on a carrier” is a reagent used for immobilizing driver DNA. . Specifically, those described in the method for concentrating trace genes can be preferably used. In addition, regarding the configuration or form of the kit, a technique known per se may be used.
    The present invention also provides an apparatus for concentrating trace genes in a DNA sample. More specifically, (a) means for making DNA in a mixed solution of target DNA and labeled driver DNA single-stranded, (b) means for performing hybridization, and (c) labeling of driver DNA. A means for removing the double-stranded DNA formed by the driver DNA and the target DNA, and (d) a DNA solution from which the double-stranded DNA obtained in (c) is removed, instead of the target DNA ( A trace gene concentrating apparatus characterized by including means for repeating the means of b) and (c). About each means, it is the same as that of the description regarding the concentration method of the said trace gene. Moreover, the combination of each means may follow a method known per se. (A) means for cleaving DNA in each of the driver DNA fraction and the target DNA fraction so that the molecular weight of the driver DNA is lower than that of the target DNA, (b) means for labeling the driver DNA, or (C) A means for attaching a linker / adapter to the target DNA may be further combined.
In another aspect of the present invention, there is provided an apparatus for concentrating trace genes in a DNA sample by (a) means for immobilizing a single-stranded driver DNA on a carrier, and (b) immobilizing the single-stranded driver DNA. A means for performing hybridization by contacting or mixing the carrier with a single-stranded target DNA solution; and (c) separating the carrier DNA and the target DNA by separating the carrier DNA and the solution. And (d) a means for repeating the means of (b) and (c) using the target DNA solution obtained in (c) instead of the target DNA solution. Examples include a gene concentrator. About each means, it is the same as that of the description regarding the concentration method of the said trace gene. Moreover, the combination of each means may follow a method known per se. Furthermore, (a) means for cleaving DNA in each of the driver DNA fraction and the target DNA fraction so that the molecular weight of the driver DNA is lower than that of the target DNA, and (b) attaching a linker / adapter to the target DNA (C) The driver DNA fraction and the target DNA fraction may each be further combined with a means for making the DNA single-stranded.
  Example
Hereinafter, the present invention will be described in detail with reference to examples, but the present invention is not limited thereto.
Example 1
E. coli B strain DNA was purchased from Sigma Co. USA and human genomic DNA was purchased from CLONTECH Laboratories Inc., USA. The DNA of Bacillus pumilus was prepared from the cells using Gen Toru-kun (trade name, manufactured by Takara Shuzo Co., Ltd.).
In this example, the following restriction enzymes, buffers, reagents and the like were used. In addition, when using each restriction enzyme or reagent, the instruction manual for the product was used.
(A) Msp I (Takara Shuzo Co., Ltd.): 10 units / μl [solvent 10 mM KPO_Four, 200 mM NaCl, 1 mM EDTA, 1 mM Dithiothreitol (DTT), 0.02% bovine serum albumin (hereinafter referred to as BSA), 50% glycerol (pH7.5))
(B) Sse 8387I (Takara Shuzo Co., Ltd.): 10 mM Tris-HCl (pH 7.5), 10 mM MgCl₂, 1mM DTT, 50mM NaCl, 100μg / ml BSA
(C) TE: 10 mM Tris-HCl, pH 7.5
(D) 10 x M: 100 mM Tris-HCl, pH 7.5, 100 mM MgCl₂, 10mM DTT, 500mM NaCl
(E) 10 x Mirus Labeling Buffer A (Mirus): 200 mM MOPS, pH 7.5
(F) 20 x SSC: 3M NaCl, 0.3M Na_Three-citrate
(G) Formamide: 1/10 volume (weight / volume) of AG501-x8, 20-50 mesh, fully regenerated (BioRad Lab. USA) was added and rotated and mixed overnight.
(H) W x B: 5 mM Tris-HCl, 0.5 mM EDTA, 1 M NaCl
(I) Dynabeads (Dynabeads M-280 Streptavidin, Japan Dynal KK): 1 ml of Dynadyad suspension was separated into Dynabead and liquid phase using Dynal MPC, and Dynabeads were again tRNA (20 μg / ml). ) Suspend in containing W x B, separate beads and liquid phase with Dynal MPC. This was repeated once more. Finally, the Dynabeads were suspended in WxB containing tRNA (20 μg / ml), and the required amount of suspension was dispensed into test tubes.
  [Operation 1 Preparation of DNA mixture]
80 μg of E. coli DNA and 8 ng of Bacillus subtilis DNA were dissolved in 440 μl of TE to obtain solution A.
80 μg of human DNA, 80 ng of E. coli DNA, and 0.8 ng of Bacillus subtilis DNA were dissolved in 700 μl of TE to obtain solution B.
  [Operation 2 Preparation of driver DNA (DNA fragmentation and biotinylation)]
To A solution containing 75 μg of E. coli DNA and 7.5 ng of Bacillus subtilis DNA, add 120 μl of 10 × B, 600 units of Msp I and water to 1.2 ml and incubate at 37 ° C. for 2 hours to cleave the DNA. Subsequently, Msp I was inactivated by treatment at 60 ° C. for 15 minutes. The reaction solution was divided into three equal parts, and the DNA fragments were ethanol precipitated and collected by centrifugation (hereinafter referred to as E / B mixed DNA fragments).
Add 75 μg of human DNA, 75 ng of E. coli DNA, 0.75 ng of Bacillus subtilis DNA to solution B, add 120 μl of 10 × B, 600 units of Msp I, water to 1.2 ml and incubate at 37 ° C. for 2 hours. The DNA was cleaved, and then Msp I was inactivated by treatment at 60 ° C. for 15 minutes. The reaction solution was divided into three equal parts, and the DNA fragments were ethanol precipitated and collected by centrifugation (hereinafter referred to as H / E / B mixed DNA fragments).
Dissolve the precipitate in each tube (each containing 1/3 equivalent of mixed DNA fragments) in 200 µl water + 25 µl 10 x Mirus Labeling Buffer A, add 25 µl Mirus Label IT reagent, and react at 37 ° C for 2 hours. DNA was labeled with biotin. 25 μl of 5 M NaCl and 550 μl of ethanol were added and stored at −20 ° C. (hereinafter referred to as biotinylated E / B mix DNA fragment as E / B driver DNA, biotinylated H / E / B mix DNA fragment as H / E / B driver DNA).
  [Operation 3 Target DNA preparation (DNA fragmentation)]
Add 10 µl of 10 x M, 10 µl of 0.1% BSA, 20.4 units of Sse 8387I and water to solution A containing 2.5 µg of Escherichia coli DNA and 0.25 ng of Bacillus subtilis DNA, and incubate at 37 ° C for 2 hours. The DNA was cleaved, and then Sse 8387I was inactivated by treatment at 60 ° C. for 15 minutes. 20 μg of tRNA, 10 μl of 3M sodium acetate, and 220 μl of ethanol were added and stored at −20 ° C. (hereinafter, this fragmented DNA is referred to as E / B target DNA).
To B solution containing 2.5 μg human DNA, 2.5 ng E. coli DNA, 0.025 ng Bacillus subtilis DNA, 10 μl of 10 × M, 10 μl of 0.1% BSA, 20.4 units of Sse 8387I and water were added to make 100 μl at 37 ° C. The DNA was cleaved by incubating for 2 hours, and then Sse 8387I was inactivated by treatment at 60 ° C. for 15 minutes. 20 μg of tRNA, 10 μl of 3M sodium acetate, and 220 μl of ethanol were added and stored at −20 ° C. (hereinafter, this fragmented DNA is referred to as H / E / B target DNA).
  [Operation 4 Hybridization]
The test tube containing the ethanol precipitate of E / B target DNA was centrifuged at 15,000 rpm × 20 minutes, and the supernatant was discarded. To the same tube, 400 μl of an E / B driver DNA ethanol precipitation suspension was added and centrifuged at 15,000 rpm for 20 minutes. The supernatant was discarded, and a suspension of 425 μl of E / B driver DNA ethanol precipitate was added and centrifuged, and the supernatant was discarded. Dissolve the final precipitate in 9.28 μl water + 0.96 μl 3N NaOH at room temperature, quench on ice, add 0.96 μl 3N HCl + 50 mM Tris-HCl, pH 7.2, then 8 μl 20 x SSC, 20.8 μl formamide And mixed well. One drop of mineral oil was dropped, sealed with a lid, and kept at 37 ° C. to allow the hybridization reaction to proceed.
The target DNA of H / E / B and the driver DNA of H / E / B were treated in the same manner to advance the hybridization reaction.
N⁰Indicates a DNA solution in which the hybrid / non-hybrid operation was performed N times.
After 24 hours, 20 μl of the hybridization reaction was removed and added to a test tube containing 1 ml equivalent of Dynabead suspension. Remaining (1⁰And 2⁰) Was kept warm. The test tube was frequently mixed for 50 minutes while being kept warm in a 43 ° C. warm bath. Apply to Dynal MPC, leave it for a few minutes, divide the liquid phase into two equal parts, add each to 1 ml of chilled ethanol at −20 ° C., and leave it in a −80 ° C. freezer for 35 minutes. A precipitate was obtained by centrifugation at 15,000 rpm for 20 minutes. Precipitate in one test tube (A)80 μ l Water of +10 μ l of 3N NaOHThe whole amount was transferred to the other test tube (B) to dissolve the precipitate. On the other hand, 1/3 equivalent of driver DNA was centrifuged under the same conditions to separate the precipitate,80 μ l Water of +10 μ l of 3N NaOHThe test tube (A) was washed with 45 μl thereof, and the entire amount was washed into (B). After adding 15 μl of 3N HCl + 50 mM Tris pH 7.2 to (B), 300 μl of −20 ° C. ethanol was added and mixed, and left in a −80 ° C. freezer for 30 minutes or more. Into the precipitate obtained by centrifugation4.64 μ l Water of +0.48 μ l 3N NaOHAdd 0.45 μl of 3N HCl + 50 mM Tris-HCl, pH 7.2, 4 μl of 20 x SSC, 10.4 μl of formamide, and overlay with 1 drop of mineral oil. And kept at 37 ° C.
After 20 hours, 40 μl of tRNA (20 μg / ml) -containing W × B was added and mixed. Mineral oil was removed as much as possible, chloroform saturated with TE was added, and chloroform extraction was performed according to a conventional method. The aqueous phase was made up to 1 ml with WxB containing tRNA (20 μg / ml) and added to a test tube containing 1 ml equivalent of washed Dynabeads. The mixture was incubated at 43 ° C for 50 minutes, and mixed every 1 to 2 minutes. Apply to Dynal MPC, leave it for a few minutes, divide the liquid phase into 2 ml and add each to a test tube containing 1 ml of ethanol cooled to -20 ° C, leave it in a -80 ° C freezer for 35 minutes or more, and then 15,000 rpm x 20 A precipitate was obtained by centrifugation for minutes. Dissolve the precipitate in 50 μl water + 5 μl 3N NaOH each at room temperature, add to one driver DNA precipitate, add 10 μl 3N HCl + 50 mM Tris pH 7.2, then 220 μl ethanol at −20 ° C. And left in a freezer at −80 ° C. for 50 minutes or longer. To the precipitate obtained by centrifugation,4.64 μ l Water of +0.48 μ l 3N NaOHAdd 0.45 μl of 3N HCl + 50 mM Tris-HCl, pH 7.2, 4 μl of 20 x SSC, 10.4 μl of formamide, and layer with 1 drop of mineral oil. And kept at 37 ° C.
After 213 hours and 50 minutes, take 10 μl, add 40 μl of tRNA (50 μg / ml) -containing W x B, remove mineral oil as described above, transfer to a test tube containing 500 μl equivalent of washed Dynabeads, It was attached to a rotator and rotated and mixed overnight at room temperature. The liquid phase was separated by Dynal MPC and treated with 0.05 ml equivalent of washed Dynabeads to obtain the final liquid phase, from which DNA was recovered by ethanol precipitation (3⁰).
Meanwhile, (1⁰And 2⁰10 μl was taken out after 119 hours and 5 minutes. The rest was kept warm. 10 μl taken out was treated with 0.5 ml equivalent of washed Dynabeads and recovered as an ethanol precipitate together with 1/6 equivalent of driver DNA as described above. 9.28 to precipitateμ l Water of +0.96 μ l 3N NaOHAdd 0.95 μl of 3N HCl + 50 mM Tris-HCl, pH 7.2, 8 μl of 20 x SSC, 20.8 μl of formamide, and overlay with 1 drop of mineral oil. Then, it was kept at 37 ° C. for 87 hours and 10 minutes. After demineralized oil, the liquid phase obtained by treatment with 1 ml equivalent of washed dynabeads was further treated with 0.1 ml equivalent of washed dynabeads to obtain the final liquid phase, from which nucleic acid was recovered by ethanol precipitation (2⁰).
The remainder of the incubation was kept at 37 ° C. for a total of 289 hours and 20 minutes, and then similarly treated with 0.5 ml and then 0.05 ml of washed dynabeads to obtain the final liquid phase, from which nucleic acid was recovered by ethanol precipitation (1⁰).
Finally, 0.5 μl etatinate and 20 μl 0.3N NaOH are added to all recovered nucleic acid precipitates and treated at 37 ° C. for 1.5 hours to decompose and remove excess tRNA. 20 μl 0.3N HCl + 5 mM Tris- After adding HCl, pH 7.2, DNA was collected by ethanol precipitation and dissolved in TE.
The flow of the above experiment was displayed (FIG. 3). In addition, the heat retention time of each fraction and the Cot calculated therefrom are summarized in the following table.
  Table 1

[Test Example 1 Search for Genes in Each Nucleic Acid Fraction]
Dissolve the precipitate in TE, add a portion of the solution and add 25 μl PCR reaction solution (2.5 μl 10 x PCR Buffer, 2 μl dNTP, 0.25 μl rTaq, 1 μl primer set, template DNA, H₂Add O to make 25 μl), add a drop of mineral oil, seal, perform 35 cycles of 94 ° C for 1 minute, 60 ° C for 1 minute, 72 ° C for 2 minutes, and leave at 72 ° C for 8 minutes. The temperature was lowered to 23 ° C. The primer set used was a mixture of 0.5 μl of Ef and Er (for E. coli gene), Bf and Br (for Bacillus subtilis gene), and Hf and Hr (for human gene).
Each of the following oligonucleotides was dissolved in TE to make a 100 μM solution and used as the primer.
(A) E. coli: ompA ecompa.gb_bal, 1-2721 The following part of CDS 172-669 was used as a primer.
Ef 1102-1119: 5 'TCCGAAAGATAACACCTG 3' (SEQ ID NO: 1)
Er 1892-1908: 5 'GGGATACCTTTGGAGAT 3' (SEQ ID NO: 2)
The amplified product by PCR is 807 base pairs.
(B) Bacillus subtilis: xynA bpxyna.gb_bal, 1-1070 The following part of CDS 61-747 is used as a primer.
Bf 243-260: 5 'ATTTAGTGCAGGCTGGAA 3' (SEQ ID NO: 3)
Br 650-672: 5 'CGTTTCATACATTTTCCCCATTG 3' (SEQ ID NO: 4)
The amplified product by PCR is 430 base pairs.
(C) Human: The following portions of IL5h j03478.gb_pr2, 1-3230 were used as primers.
Hf 1600-1629: 5 'ACTTTTTGAAAATTTTATCTTAATATGTGG 3' (SEQ ID NO: 5)
Hr 1981-2007: 5 'TGGCCGTCAATGTATTTCTTTATTAAG 3' (SEQ ID NO: 6)
The amplification product by PCR is 408 base pairs.
The following solutions were used as other solutions.
(I) 10 x PCR Buffer: 100 mM Tris-HCl, pH 8.3 +500 mM KCl + 15 mM MgCl₂
(Ii) dNTP mix: dATP + dGTP + dCTP + dTTP 2.5mM each
(Iii) Taq: 5 units / μl (Solvent: 20 mM Tris-HCl + 100 mM KCl + 0.1 mM EDTA + 1 mM DTT + 0.5% Tween 20 + 0.5% Nonidet P-40 + 50% Glycerol (Glycerol))
(Iv) DNA template
[Result 1]
As shown in FIG. 4A, the bands of the PCR products of the E. coli DNA and Bacillus subtilis gene in the E / B DNA mixture well reflected the amount of each DNA. This is performed once in hybridization (1⁰When applied, the amounts of E. coli and Bacillus subtilis genes were almost the same. 2 more times (2⁰), 3 times (3⁰), E. coli gene << Bacillus subtilis gene (Fig. 5A). That is, the Escherichia coli DNA that occupied the majority was preferentially removed. This indicated that the relative enrichment far exceeded 10,000 times.
As seen in FIG. 4B, the bands of the PCR product of the human gene, E. coli gene, and Bacillus subtilis gene in the H / E / B DNA mixture well reflected the amount of each DNA. Since the Bacillus subtilis gene is very small in the DNA mixture, there are more non-specific bands compared to the case where only Bacillus subtilis DNA is present in a very small amount, and the specific 430 base pair band is Slightly observed. This is hybridized 3 times (3⁰), It was found that the E. coli gene was significantly enriched compared to the human gene (FIG. 5B). By comparison with the control PCR (FIG. 4B), the human gene was several tens of times and the E. coli gene was 5 to 10 times, so it was calculated that the human gene was concentrated several hundred times. As for the Bacillus subtilis gene, the non-specific band disappears, and the specific band can be clearly identified, and it can be seen that significant enrichment was achieved compared to the contaminated DNA. At this time, the fact that the concentration is not particularly remarkable as compared with the E. coli gene can be interpreted as supporting that the human DNA that was initially present in excess was specifically removed.
Example 2
Sulfolobus shibatae, a thermoacidophilic bacterium, is selected as a rare microorganism, S. shibatae DNA and E. coli DNA are mixed, and the same experiment as in Example 1 is performed to selectively remove E. coli DNA. As a result, it was confirmed that S. shibatae DNA, which is present only in a thousandth of the E. coli DNA, was relatively concentrated. DNA of S. shibatae was prepared from the cells using Gen Toru-kun (trade name, manufactured by Takara Shuzo Co., Ltd.).
In Example 2, the same restriction enzymes, buffers, reagents and the like as in Example 1 were used, but the following were used as those not described in Example 1.
(a) 10 x Mirus Labeling Buffer A (Mirus): 200 mM MOPS, pH 7.5
(b) Glycogen: 20mg / ml
(c) MAGNOTEX-SA (Takara Shuzo Co., Ltd.): Hybridized DNA was removed using the MAGNOTEX-SA kit by the method described in the kit manual. That is, 50 μl (1 mg) of MAGNOTEX-SA was put in a 1.5 ml tube, and the tube was left on a magnetic stand for 1 minute, and then the supernatant was removed. The biotin-labeled DNA solution and an equal amount of 2 x Binding Buffer attached to this kit were mixed, added to the tube, mixed, and allowed to stand at room temperature for 10 minutes. After leaving the tube on a magnetic stand for 1 minute, the supernatant was collected. Further, 200 μl of 1 × Binding Buffer was added to MAGNOTEX-SA for washing, and washing was repeated once more. After mixing, the tube was left on a magnetic stand for 1 minute, and the supernatant was collected.
  [Operation 1 Preparation of DNA mixture]
80 μg of E. coli DNA and 80 ng of S. shibatae DNA were dissolved in 440 μl of TE to obtain solution A.
  [Operation 2 Preparation of driver DNA (DNA fragmentation and biotinylation)]
To A solution containing 75 μg E. coli DNA and 75 ng S. shibatae DNA, add 120 μl of 10 × B, 600 units of Msp I and water to 1.2 ml, and incubate at 37 ° C. for 2 hours to cleave the DNA. Subsequently, MspI was inactivated by treatment at 60 ° C. for 15 minutes. The reaction solution was divided into three equal parts, and the DNA fragments were ethanol precipitated and collected by centrifugation (hereinafter referred to as E / S mixed DNA fragments).
Dissolve the precipitate in each tube (each containing 1/3 equivalent of E / S mix DNA fragment) in 200 μl water + 25 μl 10 x Mirus Labeling Buffer A, add 25 μl Mirus Label IT reagent, and add to 37 ° C. For 2 hours, and the DNA was labeled with biotin. 25 μl of 5M NaCl and 550 μl of ethanol were added and stored at −20 ° C.
  [Operation 3 Preparation of Target DNA (Partial Decomposition of DNA)]
To solution A containing 2.5 μg of S. shibatae DNA, add 10 μl of 10 × M, 10 μl of 0.1% BSA, 20.4 units of Sse 8387I and water to 100 μl, and incubate at 37 ° C. for 2 hours to cleave the DNA. Subsequently, Sse 8387I was inactivated by treatment at 60 ° C. for 15 minutes. 10 μg of NaOAc, 220 μl of ethanol, and 1 μl of glycogen were added and stored at −20 ° C. (hereinafter referred to as E / S target DNA fragment).
  [Operation 4 Hybridization]
The test tube containing the ethanol precipitate of E / S target DNA was centrifuged at 15000 rpm × 20 minutes, and the supernatant was discarded. 400 μl of an ethanol precipitation suspension of E / S driver DNA was added to the same tube and centrifuged at 15000 rpm × 20 minutes. The supernatant was discarded, and 425 μl of an E / S driver DNA ethanol suspension was added and centrifuged. The supernatant was discarded. Dissolve the final precipitate in 9.28 μl water + 0.96 μl 3N NaOH at room temperature, quench on ice, add 0.96 μl 3N HCl + 50 mM Tris-HCl, pH 7.2, then add 8 μl 20 × SSC, 20.8 μl formamide. Mix well, add this to 0⁰It was. One drop of mineral oil was dropped, sealed with a lid, and kept at 37 ° C. to allow the hybridization reaction to proceed. After 24 hours, 10 μl of the hybridization reaction solution was taken out, and biotin-labeled DNA was removed with 1 mg of MAGNOTEX-SA. 20 μl of NaOAc, 500 μl of −20 ° C. ethanol, and 1 μl of glycogen are added to the collected supernatant, and a precipitate is obtained by centrifugation at 15000 rpm × 15 minutes.⁰It was.
In addition, 10 μl of the remaining hybridization reaction solution (2⁰), 20 μl (3⁰) In each case, biotin-labeled DNA was removed by MAGNOTEX-SA. 20 μl of NaOAc, 500 μl of −20 ° C. ethanol and 1 μl of glycogen were added to the collected supernatant, and a precipitate was obtained by centrifugation at 15000 rpm × 15 minutes (2⁰Precipitation: A, 3⁰Precipitation: B). Meanwhile, 1/3 equivalent (2⁰) And 1 bottle (3⁰The driver DNA of) is centrifuged under the same conditions to separate the precipitate,80 μl of water +10 μl 3N NaOHThen, A and B were dissolved in the total amount. After adding 15 μl of 3N HCl + 50 mM Tris-HCl pH 7.2, 300 μl of −20 ° C. ethanol was added and mixed, and the mixture was left in a −80 ° C. refrigerator for 30 minutes or more. Into the precipitate obtained by centrifugation4.64 μl of water+0.48μl 3N NaOHAdd 0.45 μl 3N HCl + 50 mM Tris-HCl pH 7.2, 4 μl 20 x SSC, 10.4 μl formamide and layer one drop of mineral oil. And kept at 37 ° C. After 24 hours, 10 μl of the hybridization reaction solution is removed from the test tube of A, and the target DNA is recovered by the same procedure as above.⁰It was.
For B, biotin-labeled DNA was removed in the same manner, the supernatant was collected, and a precipitate was obtained by centrifugation at 15000 rpm for 15 minutes (3⁰Precipitation: C). Centrifuge 1/3 equivalent of driver DNA to separate the precipitate,50 μl of water +10 μl 3N NaOHThen, C was dissolved in the whole amount. 10 μl of 3N HCl + 50 mM Tris pH 7.2 was added, 300 μl of −20 ° C. ethanol was added and mixed, and the mixture was left in a −80 ° C. refrigerator for 50 minutes or more. Into the precipitate obtained by centrifugation4.64 μl of water +0.48 μl 3N NaOHAdd 0.45 μl 3N HCl + 50 mM Tris-HCl pH 7.2, 4 μl 20 x SSC, 10.4 μl formamide and layer one drop of mineral oil. And kept at 37 ° C. After 24 hours, 10 μl of the hybridization reaction solution is removed from the C tube, and the target DNA is recovered by the same procedure.⁰It was. Finally, all recovered DNA precipitates were dissolved in 50 μl of 1 × TE.
  [Test Example 1 Search for Genes in DNA Fraction]
25 μl PCR reaction (2.5 μl 10 x PCR Buffer, 2 μl dNTP, 0.25 μl rTaq, 1 μl primer set, 3 μl template DNA, H₂Prepare 25 μl by adding O) and perform 35 cycles of PCR at 94 ° C for 1 minute, 60 ° C for 1 minute, 72 ° C for 2 minutes with Thermal cycler (Takara Shuzo Co., Ltd.) and leave at 72 ° C for 8 minutes. The temperature was lowered to 4 ° C. As primer sets, 0.5 μl of Ef and Er (for E. coli gene) and Sf and Sr (for S. shibatae gene) were used, respectively.
Each of the following oligonucleotides was dissolved in TE to make a 100 μM solution, which was used as the primer.
(A) Escherichia coli: The following part of the ompA gene (ecompa.gb_bal) was used as a primer.
Ef2 5'-TCCGAAAGATAACACCTG-3 '(SEQ ID NO: 7)
Er2 5'-GGGATACCTTTGGAGAT-3 '(SEQ ID NO: 8)
The amplified product by PCR is 807 base pairs.
(B) S. shibatae: The following part of the esterase gene (EstI) was used as a primer.
Sf 5'-ATGCCCCTAGATCCTCGAATC-3 '(SEQ ID NO: 9)
Sr 5'-TCAACTTTTATCATAAAATGTACG-3 '(SEQ ID NO: 10)
The amplification product by PCR becomes 918 base pairs.
The following solutions were used as other solutions.
(I) 10 x PCR Buffer: 100 mM Tris-HCl, pH 8.3 + 500 mM KCl + 15 mM MgCl₂
(Ii) dNTPmix: dATP + dGTP + dCTP + dTTP 2.5 mM each
(Iii) Taq: 5 units / μl (solvent: 20 mM Tris-HCl + 100 mM KCl + 0.1 mM EDTA + 1 mM DTT + 0.5% Tween20 + 0.5% Nonidet P-40 + 50% glycerol)
(Iv) DNA template
  [Test Example 2 Quantification by Real-Time PCR]
In order to analyze the abundance ratio of each gene in the DNA mixture, the state of amplification for each cycle was analyzed by real-time PCR. Since it is impossible to analyze the abundance ratio of each gene using the PCR method described above, the abundance ratio of the E. coli gene and the S. shibatae gene is analyzed using the LightCycler Quick System 330 (Roche Diagnostics). did.
The PCR reaction solution composition is 5 μl template DNA, 2 μl LightCycler-DNA master SYBR GreenI, 1 μl primer set, 1.6 μl 25 mM MgCl₂At a final concentration of 3 mM, water was added to 20 μl.
Each of the following oligonucleotides was dissolved in TE to make a 10 μM solution and used as the primer.
(A) Escherichia coli: The following part of the ompA gene (ecompa.gb_bal) was used as a primer.
Ef2 5'-TCCGAAAGATAACACCTG-3 '(SEQ ID NO: 7)
Er2 5'-GGGATACCTTTGGAGAT-3 '(SEQ ID NO: 8)
The amplified product by PCR is 807 base pairs.
(B) S. shibatae: The following part of the esterase gene (EstI) was used as a primer.
Sf 5'-ATGCCCCTAGATCCTCGAATC-3 '(SEQ ID NO: 9)
Sr 5'-TCAACTTTTATCATAAAATGTACG-3 '(SEQ ID NO: 10)
The amplification product by PCR becomes 918 base pairs.
[Result 2]
As shown in FIG. 7, the E. coli gene band gradually decreased as the concentration increased.⁰It can be seen that it has been completely removed. On the other hand, the S.shibatae gene can be confirmed to be almost the same amount throughout the series of steps (note that the primer on the low molecular weight side is a dimer). The reason why there are many non-specific bands as a whole is thought to be because the target DNA was partially digested with Sau3A1.
E. The amplification curve of coli DNA shows that target DNA is 18.2 cycles, 1⁰20.74 cycles of DNA, 2⁰23.31 cycles of DNA, 3⁰The DNA has entered the exponential growth phase at 23.80 cycles, which means that the target DNA has the largest number of copies, and 1⁰, 2⁰, 3⁰It can be seen that E. coli DNA, which occupied the majority, was preferentially removed.
On the other hand, the amplification curve of S. shibatae DNA is the target DNA, 1⁰19.22 cycles of DNA, 2⁰20.58 cycles of DNA, 3⁰Both DNA have 20.65 cycles, 1⁰→ 2⁰However, the abundance ratio has hardly changed even after a series of enrichment processes compared to the rate of E. coli. Moreover, E. coli DNA and S. shibatae DNA became substantially the same amount by two times of hybridization, and it became E. coli DNA << S.shibatae DNA by three times of hybridization. From the above results, E. coli DNA shown in the electrophoresis results of FIG. 7 is selectively removed by analyzing the abundance ratio of each gene in the DNA mixture using the real-time PCR method. It was confirmed quantitatively.
Industrial applicability
By using the trace gene enrichment method, trace gene enrichment kit, or trace gene enrichment apparatus according to the present invention, it is possible to concentrate trace genes in a DNA sample until the amount is larger than that of a large quantity of genes. Moreover, it is not necessary to know a large amount of genes to be removed from the DNA sample. Therefore, there is an advantage that DNA or the like extracted from a natural sample such as soil, lake water or river water can be used as a sample.
By concentrating trace genes in this way, the total number of clones in the gene library to be screened for cloning the genes can be greatly reduced. The result is a significant reduction in human labor, costs and time. Therefore, useful genes such as genes of enzymes important for chemical industry, lead compounds of pharmaceuticals and agricultural chemicals, and synthetic genes of antibiotics can be obtained more easily and more quickly than conventional methods. In addition, a cell surface antigen gene is obtained using a method for concentrating trace genes according to the present invention, an antibody is produced using the gene or gene information, and a cell or microorganism having the antigen is identified. In addition, the cells or microorganisms can be isolated using a cell sorter such as FACS. Furthermore, a new useful gene can be obtained by analyzing the base sequence of the whole genome of the isolated cell or microorganism. In addition, by concentrating the genes of microorganisms that are parasitic or infecting animals and plants, including humans, using the method or apparatus according to the present invention, it is possible to identify parasitic and infectious microorganisms, and further clarify the functions of the genes. A wide variety of applications are possible, such as making it possible to use them.
[Sequence Listing]

[Brief description of the drawings]
Figure 1 shows the genome size and Cot_1/2Shows the relationship. More specifically, it shows the re-formation of various double-stranded nucleic acids. The genome size is shown as a nucleotide pair at the top of the figure (Neochemistry Laboratory, Nucleic Acid I, page 200).
FIG. 2 shows the reformation of calf thymus DNA. Δ (open triangle) represents a sample with a calf thymus DNA concentration of 2 μg / ml, ● represents a sample with a calf thymus DNA concentration of 10 μg / ml, and ○ represents a calf thymus DNA concentration of 600 μg / ml. represents a sample of ml, Δ (filled triangle) represents a sample with a calf thymus DNA concentration of 8,600 μg / ml, and + represents a calf thymus of 8,600 μg / ml with 43 μg / ml of radiolabeled E. coli DNA as an internal standard. Represents a sample to which DNA has been added (New Chemistry Laboratory, Nucleic Acid I, page 199).
FIG. 3 shows the operation flow of the embodiment.
FIG. 4 (A) shows that 4 μl of the E / B target DNA Sse8387I-treated fraction was amplified by PCR, and 10 μl was subjected to 3% agarose gel electrophoresis at 100 volts for 40 minutes in the usual manner. The electrophoresis gel when stained with bromide is shown. Lane 1: Escherichia coli gene, Lane 2: Bacillus subtilis gene, Lane 3: Yeast gene (reference), Lane 4: Size marker DNA (100, 200, 300, 400, 500, 600, 700, 800, 900, 1 from the bottom) , 1,000, 1,200, 1,500 base pairs.
FIG. 4 (B) shows that 4 μl of the H / E / B target DNA Sse8387I-treated fraction was amplified by PCR, 10 μl was subjected to 3% agarose gel electrophoresis by a conventional method at 100 volts for 40 minutes, The electrophoresis gel when stained with ethidium bromide is shown. Lane 1: Size marker DNA, Lane 2: Human gene, Lane 3: E. coli gene, Lane 4: Bacillus subtilis gene
Fig. 5 (A) shows the result of dissolving the alkali-treated and ethanol-precipitated nucleic acid in 18 µl water + 1 µl TE, adding 10xPCR Buffer, Bx10, dNTP, rTaq, and primer set to make 25 µl of the reaction solution. An electrophoresis gel is shown in which a portion of is subjected to 3% agarose gel electrophoresis at 100 volts for 40 minutes in the usual manner and stained with ethidium bromide. Lanes 1, 2, 4, 5, 7, and 8 used 3 μL, and Lanes 3 and 6 used a portion of 9 μL. Lane 1: 1⁰E. coli gene, lane 2: 1⁰Bacillus subtilis gene, lane 3: 2⁰E. coli gene, lane 4: 2⁰E. coli gene, lane 5: 2⁰Bacillus subtilis gene, lane 6: 3⁰E. coli gene, lane 7: 3⁰E. coli gene, lane 8: 3⁰Bacillus subtilis gene, Lane 9: Size marker DNA
FIG. 5 (B) shows a solution of alkali-treated and ethanol-precipitated nucleic acid dissolved in 18 μl water + 1 μl TE, 10 × PCR Buffer, Bx10, dNTP, rTaq, and primer set are added to make a 25 μl reaction solution. An electrophoresis gel is shown when a part of the reaction solution is subjected to 3% agarose gel electrophoresis at 100 volts for 40 minutes in the usual manner and stained with ethidium bromide. Lane 1: size marker DNA, lane 2: 3⁰Human gene, lane 3: 3⁰E. coli gene, lane 4: 3⁰Bacillus subtilis gene
FIG. 6 shows the flow of operations in one embodiment of the trace gene enrichment method of the present invention. The dotted line shows the flow of operation when detecting the removal state of a large amount of genes.
FIG. 7 shows the results of Test Example 1 of Example 2. Target DNA (0⁰) 1⁰2⁰3⁰Each gene was amplified by PCR. 8 μl was subjected to electrophoresis with 0.8% agarose (manufactured by Nippon Gene Co., Ltd.) at 100 V for 30 minutes and stained with ethidium bromide. A λ-Hind III digest was used as a size marker. Lane 1: Size marker DNA (λ-Hind III), Lane 2: 0⁰E. coli gene, lane 3: 0⁰S. shibatae gene, lane 4: 1⁰E. coli gene, lane 5: 1⁰S. shibatae gene, lane 6: 2⁰E. coli gene, lane 7: 2⁰S. shibatae gene, lane 8: 3⁰E. coli gene, lane 9: 3⁰S. shibatae gene, lane 10: PCR reaction solution containing no E. coli template DNA, lane 11: S. p. PCR reaction solution containing no shibatae template DNA

Claims (13)

Concentration of genes present in trace amounts, characterized in that DNA samples containing genes present in trace amounts and genes present in large amounts are subjected to the following operations to separate genes present in trace amounts from genes present in large amounts Method.
(A) Divide the DNA sample into 2 minutes. One DNA sample is a driver DNA fraction and the other DNA sample is a target DNA fraction.
(B) The target DNA and the driver DNA are mixed, and the DNA in the mixed solution is made into a single strand. Alternatively, the target DNA and driver DNA are mixed into a single strand.
(C) Hybridization is performed, and the double-stranded DNA formed by the driver DNA and the target DNA is removed from the mixed solution.
(D) The operations of (b) and (c) are performed one or more times using the DNA solution obtained by removing the double-stranded DNA obtained in (c) instead of the target DNA. Concentration of genes present in trace amounts, characterized in that DNA samples containing genes present in trace amounts and genes present in large amounts are subjected to the following operations to separate genes present in trace amounts from genes present in large amounts Method.
(A) Divide the DNA sample into 2 minutes. One DNA sample is a driver DNA fraction and the other DNA sample is a target DNA fraction.
(B) The DNA is cleaved in each of the driver DNA fraction and the target DNA fraction. However, at this time, the molecular weight of the driver DNA is set to be lower than that of the target DNA.
(C) Label the driver DNA.
(D) The target DNA and an excessive amount of labeled driver DNA are mixed, the DNA in the mixed solution is made into a single strand, and then hybridization is performed.
(E) The double-stranded DNA formed by the driver DNA and the target DNA is removed from the mixed solution by labeling the driver DNA.
(F) The operations of (d) and (e) are performed one or more times using the DNA solution obtained by removing the double-stranded DNA obtained in (e) instead of the target DNA. The method for concentrating a gene present in a trace amount according to claim 2, further comprising attaching a linker adapter to the target DNA in (c). The ratio d / t of a mixed amount of driver DNA (d) and mixing the amount of target DNA and (t) is a trace amount of any one of claims 1 to 3, wherein the 1000 or less than 1 Of enriching genes present in The method for concentrating a gene present in a trace amount according to any one of claims 2 to 4 , wherein the driver DNA is labeled with biotin, digoxin, fluorescein or rhodamine. The average chain length of the driver DNA is 200-300 bp, genes present in a trace amount of any one of claims 2-5 having an average chain length of the target DNA is characterized in that 1000 bp or more Concentration method. Cleavage of DNA is, by 4 base recognition restriction enzymes in the driver DNA fraction, the small amount of any of claims 2-6 in which the target DNA fraction characterized by being carried out by 5-8 base recognition restriction enzyme A method for enriching existing genes. The method for concentrating a gene present in a trace amount according to claim 7 , wherein the DNA is cleaved by Msp I in the driver DNA fraction and by Sse 8387I in the target DNA fraction. The method for concentrating a gene present in a trace amount according to any one of claims 2 to 6, wherein the DNA is cleaved by ultrasonic waves or mechanical shearing force. Concentration of genes present in trace amounts, characterized in that DNA samples containing genes present in trace amounts and genes present in large amounts are subjected to the following operations to separate genes present in trace amounts from genes present in large amounts Method.
(A) Divide the DNA sample into 2 minutes. One DNA sample is a driver DNA fraction and the other DNA sample is a target DNA fraction.
(B) The DNA is cleaved in each of the driver DNA fraction and the target DNA fraction. However, at this time, the molecular weight of the driver DNA is set to be lower than that of the target DNA.
(C) In each of the driver DNA fraction and the target DNA fraction, the DNA is made into a single strand.
(D) Immobilize single-stranded driver DNA on a carrier.
(E) A carrier on which a single-stranded driver DNA is immobilized is brought into contact with or mixed with a single-stranded target DNA solution to perform hybridization.
(F) By separating the carrier and the solution, the target DNA that forms a double strand with the driver DNA is removed.
(G) The operations of (e) and (f) are performed one or more times using the target DNA solution obtained in (f) instead of the target DNA solution. The method for concentrating genes according to claim 10, wherein in ( b), a linker adapter is further attached to the target DNA. A DNA sample extracted from a sample in which at least two types of microorganisms, organisms, biological tissues or cells are mixed, or a nucleic acid extracted from a living individual, biological tissue or cells and / or a DNA sample prepared from the nucleic acid The method for concentrating a gene present in a trace amount according to any one of claims 1 to 11, wherein: Gene present in trace amounts in the process according to any one of claims 1 to 11 (hereinafter, referred to as "trace genes") step of concentrating the step of trace genes obtained acquires trace genes from the concentrated DNA samples And a method for analyzing a trace gene, comprising the step of analyzing the base sequence of the trace gene.

Images